Immunomodulatory Effect of Toll-Like Receptor-3 Ligand Poly I:C on Cortical Spreading Depression

The release of inflammatory mediators following cortical spreading depression (CSD) is suggested to play a role in pathophysiology of CSD-related neurological disorders. Toll-like receptors (TLR) are master regulators of innate immune function and involved in the activation of inflammatory responses in the brain. TLR3 agonist poly I:C exerts anti-inflammatory effect and prevents cell injury in the brain. The aim of the present study was to examine the effect of systemic administration of poly I:C on the release of cytokines (TNF-α, IFN-γ, IL-4, TGF-β1, and GM-CSF) in the brain and spleen, splenic lymphocyte proliferation, expression of GAD65, GABAAα, GABAAβ as well as Hsp70, and production of dark neurons after induction of repetitive CSD in juvenile rats. Poly I:C significantly attenuated CSD-induced production of TNF-α and IFN-γ in the brain as well as TNF-α and IL-4 in the spleen. Poly I:C did not affect enhancement of splenic lymphocyte proliferation after CSD. Administration of poly I:C increased expression of GABAAα, GABAAβ as well as Hsp70 and decreased expression of GAD65 in the entorhinal cortex compared to CSD-treated tissues. In addition, poly I:C significantly prevented production of CSD-induced dark neurons. The data indicate neuroprotective and anti-inflammatory effects of TLR3 activation on CSD-induced neuroinflammation. Targeting TLR3 may provide a novel strategy for developing new treatments for CSD-related neurological disorders. © 2014, Springer Science+Business Media New York

Immunomodulatory Effect of Toll-Like Receptor-3 Ligand Poly I:C on Cortical Spreading Depression

The release of inflammatory mediators following cortical spreading depression (CSD) is suggested to play a role in pathophysiology of CSD-related neurological disorders. Toll-like receptors (TLR) are master regulators of innate immune function and involved in the activation of inflammatory responses in the brain. TLR3 agonist poly I:C exerts anti-inflammatory effect and prevents cell injury in the brain. The aim of the present study was to examine the effect of systemic administration of poly I:C on the release of cytokines (TNF-α, IFN-γ, IL-4, TGF-β1, and GM-CSF) in the brain and spleen, splenic lymphocyte proliferation, expression of GAD65, GABAAα, GABAAβ as well as Hsp70, and production of dark neurons after induction of repetitive CSD in juvenile rats. Poly I:C significantly attenuated CSD-induced production of TNF-α and IFN-γ in the brain as well as TNF-α and IL-4 in the spleen. Poly I:C did not affect enhancement of splenic lymphocyte proliferation after CSD. Administration of poly I:C increased expression of GABAAα, GABAAβ as well as Hsp70 and decreased expression of GAD65 in the entorhinal cortex compared to CSD-treated tissues. In addition, poly I:C significantly prevented production of CSD-induced dark neurons. The data indicate neuroprotective and anti-inflammatory effects of TLR3 activation on CSD-induced neuroinflammation. Targeting TLR3 may provide a novel strategy for developing new treatments for CSD-related neurological disorders. © 2014, Springer Science+Business Media New York

Linking immune-mediated damage to neurodegeneration in multiple sclerosis: could network-based MRI help?

Inflammatory demyelination characterizes the initial stages of multiple sclerosis, while progressive axonal and neuronal loss are coexisting and significantly contribute to the long-term physical and cognitive impairment. There is an unmet need for a conceptual shift from a dualistic view of multiple sclerosis pathology, involving either inflammatory demyelination or neurodegeneration, to integrative dynamic models of brain reorganization, where, glia-neuron interactions, synaptic alterations and grey matter pathology are longitudinally envisaged at the whole-brain level. Functional and structural MRI can delineate network hallmarks for relapses, remissions or disease progression, which can be linked to the pathophysiology behind inflammatory attacks, repair and neurodegeneration. Here, we aim to unify recent findings of grey matter circuits dynamics in multiple sclerosis within the framework of molecular and pathophysiological hallmarks combined with disease-related network reorganization, while highlighting advances from animal models (in vivo and ex vivo) and human clinical data (imaging and histological). We propose that MRI-based brain networks characterization is essential for better delineating ongoing pathology and elaboration of particular mechanisms that may serve for accurate modelling and prediction of disease courses throughout disease stages

Cytotoxic CD8+ T cell-neuron interactions: perforin-dependent electrical silencing precedes but is not causally linked to neuronal cell death

Cytotoxic CD8(+) T cells are considered important effector cells contributing to neuronal damage in inflammatory and degenerative CNS disorders. Using time-lapse video microscopy and two-photon imaging in combination with whole-cell patch-clamp recordings, we here show that major histocompatibility class I (MHC I)-restricted neuronal antigen presentation and T cell receptor specificity determine CD8(+) T-cell locomotion and neuronal damage in culture and hippocampal brain slices. Two separate functional consequences result from a direct cell-cell contact between antigen-presenting neurons and antigen-specific CD8(+) T cells. (1) An immediate impairment of electrical signaling in single neurons and neuronal networks occurs as a result of massive shunting of the membrane capacitance after insertion of channel-forming perforin (and probably activation of other transmembrane conductances), which is paralleled by an increase of intracellular Ca(2+) levels (within <10 min). (2) Antigen-dependent neuronal apoptosis may occur independently of perforin and members of the granzyme B cluster (within approximately 1 h), suggesting that extracellular effects can substitute for intracellular delivery of granzymes by perforin. Thus, electrical silencing is an immediate consequence of MHC I-restricted interaction of CD8(+) T cells with neurons. This mechanism is clearly perforin-dependent and precedes, but is not causally linked, to neuronal cell death

Continuous short-term structural network reorganisation beyond atrophy in patients with RRMS [Abstract]

Background and aim: Longitudinal assessment of structural brain changes is important to track the clinical course of multiple sclerosis (MS), but an exact quantification of the diffuse tissue damage is highly challenging. We aimed to identify short-term structural dynamics by measuring grey matter (GM) network connectivity patterns and comparing these with established morphological measures of GM integrity. Methods: For our prospectively designed study, we collected data from January 2013 through December 2014. In total, forty-five structural MRI datasets from relapsing-remitting MS patients in the relapse free phase of the disease (mean age: 42 ± 12.1 years; median EDSS 1.5 (0 - 2.5); mean disease duration 3.5 ± 6.5 years) were acquired using 3T MRI. Each patient was followed up every 8 weeks for 8 months and all patients were enrolled at two German university hospitals. Longitudinal brain atrophy was analyzed using SIENA (part of FSL), while FreeSurfer was used to investigate cortical thickness changes over time. GM connectivity patterns were reconstructed from cortical thickness correlation matrix between anatomical regions, as derived from the AAL atlas, and a network analysis was conducted using graph theoretical approaches. Results: Our study shows a significant longitudinal structural network reorganisation in the absence of cortical thinning and brain atrophy already over a period of 4 months. We demonstrate an increased local (clustering coefficient (F(4,41) = 3.547, p < 0.001), local efficiency (F(4,41) = 3.0874, p < 0.01)) and modular connectivity pattern (modularity (F(4,41) =2.612, p < 0.01)). Conversely a concomitant break-down of long-range connectivity occurred (assortativity (F(4,41) = 3.0654, p < 0.01) and small-world index (F(4,41) = 3.687, p < 0.001)). No regional or global atrophy signs were detected in the applied morphometric analysis. Conclusions and relevance: Our GM network analysis demonstrates a short-term increase in local connectivity and a decrease in long-range paths in MS patients in the relapse free state of the disease, in the absence of atrophy or clinical progression. Structural reorganisation patterns with co-occurrence of detrimental and adaptive reorganisation processes might be important sensitive measurable fingerprints of the disease that can be used in clinical practice

Longitudinal structural network reorganisation in early relapsing-remitting multiple sclerosis [Abstract]

Background: Multiple sclerosis (MS) is characterized by relapses and remissions indicating damage and compensatory processes occurring early in the disease. Over time, cortical pathology is highly relevant for disability, while brain networks evolve towards a disconnected organization as the disease progresses. However, it is poorly understood how and when pathology impacts cortical networks and in particular, how the network responds to damage in the very beginning of the disease. Aim: To address cortical pathology by quantifying structural connectivity patterns over 12 months in patients with early relapsing-remitting MS. Methods: Here we investigated cortical grey matter networks longitudinally as derived from structural 3 Tesla MRI in 92 patients in the initial phase of the disease (65 female / 27 male; mean age: 32.9 ± 9.9 years; mean disease duration: 12.1 ± 14.5 months) and in 101 healthy controls (59 female / 42 male; mean age: 19.7 ± 0.9 years). Longitudinal brain volume atrophy was analyzed using SIENA and cortical thickness changes were quantified using FreeSurfer. Brain networks were computed based on cortical thickness inter-regional correlations between anatomical regions and fed into graph theoretical analysis. Finally, subgroup analyses were performed between patients with “no evidence of disease activity” (NEDA) during this period and those with disease activity (EDA). Results: Over one year, increased local cortical connectivity and an emerging modular-constructed network were detected in patients - a pattern reported to be associated with adaptation, efficiency and compensation. These longitudinal dynamics were attested in both patients with NEDA and EDA, indicating continuous cortical reorganisation independent of disease activity. This local and modular cortical reorganisation was not detected in healthy controls over the same period of time and emerged beyond measureable signs of atrophy using established morphometric tools. Conclusion: Our findings demonstrate that despite initiation of neuroinflammatory damage, substantial structural adaptation processes emerge cortically in the early disease stage. This subtle reorganisation of the cortex architecture is quantifiable by structural MRI in patients with and without disease activity, suggesting a principal response of the network evolving from the onset of this chronic disease. Disclosure: The authors declare no conflict of interests

Continuous reorganisation of cortical information flow in MS patients: a longitudinal effective connectivity study [Abstract]

Background: Brain reorganisation processes are essential for the long-term outcome in patients with multiple sclerosis (MS). Effective connectivity (EC) as derived from functional MRI, can be analysed to estimate reorganisation processes and directional information flows between cortical regions. These measures could provide the missing link for modelling the long-term disease course between tissue damage and repair or adaptation. Aim: To obtain longitudinal measurements of EC and information flows in MS patients at short-term intervals focusing on the main anatomical brain regions and to investigate the link between the connectivity strength and clinical impairment. Methods: Twelve MS patients (mean age: 41.7 ± 11.5 years) underwent 3 Tesla structural and resting state functional MRI at five different time points over one year (approximately every 12 weeks). Twelve healthy subjects (mean age: 33.5 ± 9.6 years) served as controls (HC). For the analytical framework, two novel approaches for EC quantification were used. Causal Bayesian Network (CBN) and Time Domain Partial Directed Coherence (TPDC) were applied for the description of the information flows between frontal, prefrontal, temporal, occipital, and parietal lobe; cerebellum and deep grey matter nuclei (DGMN) were also analysed. Results: Specific longitudinal EC patterns have been attested in the studied regions. Information flows from DGMN, frontal, prefrontal and temporal to the other studied regions showed a continuous increase over time, whereas the directed connections from parietal and occipital lobes and from the cerebellum did not change over time as confirmed by both applied methods. No longitudinal changes of EC were attested in HC. The longitudinal connectivity increase in the prefrontal-frontal and fronto-cerebellar pathway showed a significant inverse correlation to EDSS (Expanded Disability Status Scale). Moreover, the EC change from the frontal lobe to the cerebellum showed a significant inverse correlation to patients’ fatigue score. Conclusion: Our data depicts a continuous longitudinal increase in EC in patients with MS substantiated by two novel methodological approaches. Furthermore, the dynamics of the fronto-cerebellar connections are linked to clinical impairment and possibly essential for the long-term outcome

Excitotoxic neuronal cell death during an oligodendrocyte-directed CD8+ T cell attack in the CNS gray matter

Background: Neural-antigen reactive cytotoxic CD8+ T cells contribute to neuronal dysfunction and degeneration in a variety of inflammatory CNS disorders. Facing excess numbers of target cells, CNS-invading CD8+ T cells cause neuronal cell death either via confined release of cytotoxic effector molecules towards neurons, or via spillover of cytotoxic effector molecules from 'leaky’ immunological synapses and non-confined release by CD8+ T cells themselves during serial and simultaneous killing of oligodendrocytes or astrocytes. Methods: Wild-type and T cell receptor transgenic CD8+ T cells were stimulated in vitro, their activation status was assessed by flow cytometry, and supernatant glutamate levels were determined using an enzymatic assay. Expression regulation of molecules involved in vesicular glutamate release was examined by quantitative real-time PCR, and mechanisms of non-vesicular glutamate release were studied by pharmacological blocking experiments. The impact of CD8+ T cell-mediated glutamate liberation on neuronal viability was studied in acute brain slice preparations. Results: Following T cell receptor stimulation, CD8+ T cells acquire the molecular repertoire for vesicular glutamate release: (i) they upregulate expression of glutaminase required to generate glutamate via deamination of glutamine and (ii) they upregulate expression of vesicular proton-ATPase and vesicular glutamate transporters required for filling of vesicles with glutamate. Subsequently, CD8+ T cells release glutamate in a strictly stimulus-dependent manner. Upon repetitive T cell receptor stimulation, CD25high CD8+ T effector cells exhibit higher estimated single cell glutamate release rates than CD25low CD8+ T memory cells. Moreover, glutamate liberation by oligodendrocyte-reactive CD25high CD8+ T effector cells is capable of eliciting collateral excitotoxic cell death of neurons (despite glutamate re-uptake by glia cells and neurons) in intact CNS gray matter. Conclusion: Glutamate release may represent a crucial effector pathway of neural-antigen reactive CD8+ T cells, contributing to excitotoxicity in CNS inflammation.<br

CSF markers of blood-brain barrier integrity forecast disease progression in early MS [Abstract]

Background and aim: Cortical atrophy, reflecting neuronal loss, is highly associated with long-term disability in patients with multiple sclerosis (MS). In this longitudinal study we link cerebrospinal fluid (CSF) markers of blood-brain barrier (BBB) integrity to longitudinal cortical atrophy and clinical disability. Methods: 71 relapsing-remitting multiple sclerosis (RRMS) patients (31.2 ± 9.4 (mean ± SD), 25 males) were included in this longitudinal study. CSF and serum samples were obtained from each patient at the time of first clinical event. We analyzed CSF levels of albumin (AlbCSF), immunoglobulin A (IgACSF), IgG (IgGCSF) and IgM (IgMCSF). All patients underwent MR imaging twice with the same standardized protocol (follow-up after 12 ± 1 months) at 3T (Siemens Magnetom Trio). Longitudinal changes of cortical thickness (CT) were extracted using the FreeSurfer processing stream. The Expanded Disability Status Scale (EDSS) score at follow-up was used as a clinical outcome measure to quantify clinical disability. The rate of cortical atrophy was assessed in relation to CSF variables. Results: Baseline AlbCSF and IgACSF were highly associated with the rate of cortical atrophy over one year. Significant correlations were found in the precuneus (PrC), rostral middle frontal, precentral and inferior parietal gyri of both hemispheres. The regions with the highest atrophy rates were the right PrC (R = 0.604, p < 0.001) and left fusiform gyrus (R = 0.539, p < 0.001). IgACSF and IgMCSF (IgA: 1.67 ± 0.69 mg/l vs 2.03 ± 0.71 mg/l, IgM: 9.87 ± 2.38 mg/l vs 11.5 ± 2.03 mg/l; p = 0.04 and p = 0.003, respectively) significantly differed between patients with no disability (EDSS 0 - 1.5) and those with mild to moderate disability (EDSS 2 - 6) at the second time point. Conclusion: Our data show that widespread cortical atrophy is highly associated with increased baseline CSF Albumin and IgA mirroring a permeable BBB. Patients with this BBB pattern showed higher functional disability at follow-up. These parameters could be addressed to dichotomize patients at risk of rapid disease progression

Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4(-/-)) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4(-/-) mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy